The long term goal of this proposal is to develop a strategy to interrupt both acute and reactivation toxoplasmosis. This will be done by taking advantage of gliding motility which is a unique mechanism of parasite mobility. Development of pharmacological agents that interfere with T. gondii invasion and intracellular survival will be facilitated by an understanding of the molecular mechanisms effecting gliding motility of zoites. T. gondii myosins will be the major focus of this work as they are the likely primary motor proteins. Cloning will by PCR amplification of myosin from cDNA and genomic libraries using degenerate oligonucleotide primers designed from highly conserved sequences in heterologous myosin genes. Characterization of such myosin genes may add considerable information regarding the functional and regulatory domains of the parasite myosins. Expression of parasite myosin genes will be studied in motile and immotile pathogenic life stages. Parasite-specific anti-myosin antibodies will be produced for morphologic and immunochemical analysis of myosin distribution. Manipulation of T. gondii myosin expression by mechanisms of transfection, insertional mutagenesis, or gene knockout will allow study of myosin regulation and its role in motility and invasion directly. The mechanism of agents affecting motility will be studied by observing changes in the actomyosin cytoskeleton and the resultant deficits in invasion, secretion or signal transduction. Selection of resistance mutants will allow reverse genetics approaches for discovery of the targets of agents affecting motility.